Invasive fungal infections (IFI) remain severe and underappreciated causes of disease, and associated with high morbidity and mortality worldwide. Candida albicans impacts strongly the incidence of IFI, and non-albicans Candida species have emerged as a growing global public health concern. Paracoccidioides brasiliensis is the main responsible for an endemic IFI identified in the Latin America. Additionally, Rhizopus oryzae is the most common cause of mucormycosis, a severe and often life-threatening IFI. The host immune response against IFI relies on the effector T cells. However, T cell activity is impaired in immunocompromised individuals, increasing susceptibility to IFI. Chimeric antigen receptor (CAR) technology offers a promising strategy to redirect T cells to specifically recognize and attack fungal pathogens at the site of infection. Our group developed a novel CAR, called M-CAR, capable to induce T cell activation after recognition of Candida spp., P. brasiliensis and R. oryzae. The antigen recognition domain of M-CAR is compound of a single-chain variable fragment (scFv) with specificity to α-1,6 mannose backbone of fungal mannan, a carbohydrate presents on the fungi cell wall. M-CAR has a FLAG-tag on the N-terminus, the CD8 molecule as hinge/transmembrane and CD3ζ as signaling domain. As costimulatory domain, the original M-CAR construct contained CD137 molecule.

However, in this study, we evaluated alternative costimulatory molecules in both second- and third-generation M-CARs to screen costimulatory domains able to enhance T cell activation against the target.

We generated M-CAR variants incorporating either CD28 or iCOS as coestimulatory domain, as second generation. Additionally, we developed two variants of third-generation M-CARs combining costimulatory domains along with a CD3ζ, as follows: iCOS and CD137; iCOS and CD28. The M-CAR constructs were generated using the Gibson Assembly method. Lentiviral particles containing the coding sequence of the different constructs of M-CAR were made in HEK 293T cells after transfection. These lentiviral particles were used to transduce Jurkat cell line and primary human T cells, which was isolated from peripheral blood mononuclear cells (PBMC) using Ficoll. The modified cells were co-cultured with soluble mannan obtained from S. cerevisiae, different species and morphologies of Candida, P. brasiliensis yeasts or R. oryzae spores.

All different M-CAR variants were efficiently expressed on the surface of Jurkat cells. After 24 hours of co-culture with the target fungi Jurkat cells expressing M-CAR variants exhibited increased expression of CD69, an early marker of T cell activation. This upregulation was observed in response to mannan, as well as heat-killed C. albicans and C. tropicalis hyphae, C. parapsilosis pseudohyphae, C. glabrata yeast, P. brasiliensis yeast and R. oryzae spores. Cells expressing M-CAR containing as costimulatory domains iCOS, CD28, iCOS-CD28 and iCOS-CD137 enhanced the CD69 expression, whereas the strongest activation cell came from M-CAR containing CD137 alone as costimulatory domain. Primary T cells were effectively transduced with second-generation M-CAR constructs, and preliminary data suggest that these receptors enhance IFN-γ production in response to C. albicans.

These data open the way to evaluate the fungicidal activity of human T cells expressing the M-CAR variants in response to fungal species reported in the current work.

FAPESP 2024/00060-8